Abstract
Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.
Highlights
Because the combination of Cys-819 and Cys-110 increases the sensitivity of IDE by H2O2 and GSNO, we evaluated if the combination of Cys-178 with Cys-110 has a similar effect
We found that IDE-CF-110C/178C is relatively unaffected by H2O2 and GSNO (Fig. 4B)
It is worth noting that the protection against oligomerization, rendered by Cys-178 is not complete for IDE-CF789C/966C/819C/178C because most of the enzyme eluted at position 3 and not 4 as in the control (Fig. 5H)
Summary
NEM, indicating that a different mechanism is likely involved in these physiologically relevant regulations (13). The molecular basis of IDE regulation by oxidation and nitrosylation has not been explored. Whether an oxidative burst can inhibit endogenous IDE in cell-culture based conditions has not been examined. To better understand the effect and underpinning mechanism of oxidative and nitrosylative modification of IDE, we investigated whether an oxidative burst of the microglial cell line, BV-2, can inhibit the activity of secreted IDE. We examined the biochemical effect of the ROS/RNS modification of the enzyme by fully characterizing the kinetics and the biophysical properties of the modified form of IDE.
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